This invention describes a family of affinity media for use in affinity chromatography. Specifically, this invention describes the synthesis of a new affinity medium, the p-aminophenyl-.gamma.-ester of 7-methylguanosine-5'-triphosphate coupled to Sepharose, for use in affinity chromatography. This invention also describes an improved method for the isolation of messenger ribonucleic acid capbinding protein (CBP). CBP isolated by the improved method can be obtained in higher yield, and, to the limited extent to which it has been tested, has greater activity than CBP isolated with earlier methods.
CBP is a 24 kd (kilodalton) protein which binds specifically to messenger ribonucleic acid (m-RNA). CBP has been purified by conventional methods of protein fractionation. Hellmann et al. ((1982), J. Biol. Chem. 257, 4056) used a procedure which involved saturating rabbit reticulocyte ribosome extracts with ammonium sulfate, chromatography of the precipitate on diethylaminoethyl (DEAE)--cellulose, centrifugation on sucrose gradients containing 100 and 500 mM KCl, and column chromatography on DEAE--cellulose and phosphocellulose. Trachsel et al. ((1980) Proc. Natl. Acad. Sci. U.S.A. 77, 770) utilized ammonium sulfate fractionation, centrifugation on sucrose gradients containing 100 and 500 mM KCl, phosphocellulose chromatography, affinity chromatography with a medium of eIF-3 (an initiation factor) coupled to Sepharose 4B and phospho-cellulose chromatography to purify the CBP. The speed and simplicity of the isolation and purification of cap-binding protein would be greatly improved if affinity chromatography alone could be employed. Two reports have described the synthesis of different affinity media for the purification of CBP.
Sonenberg et al. ((1979), Proc. Natl. Acad. Sci. U.S.A. 76, 4345) coupled the levulinic acid acetal of 7-methylguanosine diphosphate (m.sup.7 GDP) to Sepharose (FIG. 1, resin 1), and Rupprecht et al. ((1981), Biochemistry 20, 6570), a 7-carboxypentyl derivative of GDP (FIG. 1, resin 2). Both were capable, with limitations, of specifically purifying the 24-kd CBP. Sonenberg et al. reported that application of a 0-40% ammonium sulfate fraction of the ribosomal salt wash to resin 1 did not yield any polypeptides upon elution with m.sup.7 GDP. It was necessary to first remove eIF-3, another peptide which binds CBP, by sucrose gradient centrifugation before application of extracts to the affinity material if the CBP was to be obtained. Rupprecht et al. also reported that not all of the CBP could be eluted with m.sup.7 GDP from resin 2; a considerable amount was present in the 1.0M KCl wash which followed. Therefore, while affinity chromatography with the above media reduces the number of steps required to purify CBP, some prepurification of the ribosomal salt washes is required if non-specific binding is to be minimized.